Acridan compounds

ABSTRACT

A chemiluminescent assay method, compositions, kits and chemiluminescent acridan compounds are described which use a two-step chemiluminescent reaction process. The reaction involves an acridan compound, preferably a derivative of an N-alkylacridan-9-carboxylic acid, which undergoes a reaction with a peroxide compound, a peroxidase enzyme and an enhancer under conditions of time, temperature and pH which permit the accumulation of an intermediate compound, which is subsequently induced to produce a burst of light by raising the pH. The result is generation of very high intensity light from the reaction. The peroxidase enzyme is present alone or linked to a member of a specific binding pair in an immunoassay, DNA probe assay or other assay where the hydrolytic enzyme is bound to a reporter molecule. The method is particularly amenable to automated assays because of the separation of the incubation and light generating steps.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation-In-Part of application Ser. No.08/300,462 filed Sep. 2, 1994, which is a Continuation-In-Part ofapplicant's application Ser. Nos. 08/205,093 filed Mar. 2, 1994 and08/228,290 filed Apr. 15, 1994, now U.S. Pat. No. 5,523,212, which areContinuations-In-Part of application Ser. No. 08/061,810 filed May 17,1993, now U.S. Pat. No. 5,491,072.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to acridan compounds, particularlyN-alkylacridancarboxylic acid derivatives which allow the production oflight (chemiluminescence) by reaction with a peroxide and a peroxidase.This invention relates to stabilized chemiluminescent acridiniumcompounds, which are formed by the peroxidase-catalyzed oxidation ofacridan compounds and whose chemiluminescent reaction with peroxide canbe prevented until the desired time. This invention relates to animproved method of generating light chemically by the action of aperoxidase enzyme and an oxidant such as hydrogen peroxide with a groupof N-alkylacridancarboxylic acid derivatives in which the enzymatic stepand light producing step can be separated in time. The invention alsorelates to the use of specific enhancer substances for enhancing theamount of chemiluminescence produced from this method. The inventionalso relates to the use of this method to detect the hydrogen peroxideor peroxidase enzymes by a chemiluminescent assay. Further, theinvention relates to the use of the method to detect and quantitatevarious biological molecules which can be directly or indirectly boundto a peroxidase enzyme. For example, the method may be used to detecthaptens, antigens, proteins and antibodies by the technique ofimmunoassay, and DNA or RNA by nucleic acid hybridization assays. Themethod may additionally be used to detect enzymes which generatehydrogen peroxide such as oxidase and dehydrogenase enzymes orconjugates of such enzymes with biological molecules. Thechemiluminescent methods of the invention are particularly useful forthe detection of biological molecules in assays performed on automatedinstruments.

2. Description of Related Art

The detection and quantitation of biological molecules has beenaccomplished historically with excellent sensitivity by the use ofradiolabeled reporter molecules. Recently numerous non-radioactivemethods have been developed to avoid the hazards and inconvenience posedby these materials. Enzyme-linked detection techniques offer the bestsensitivity since the catalytic turn over of substrate to produce adetectable change in effect amplifies the detected signal. Substrateswhich generate color, fluorescence or chemiluminescence have beendeveloped, the latter achieving the best sensitivity.

Further increases in assay sensitivity will expand the range of utilityof chemiluminescence-based methods by permitting the detection ofanalytes present in smaller quantities or reducing the amount of timeand/or reagents required to perform the assay. A way to increase thespeed and sensitivity of detection in a chemiluminescent assay is tocause the light to be emitted as a short pulse of high intensity.

Horseradish peroxidase (HRP) is one of the most extensively used enzymesin enzyme-linked detection methods such as immunoassays, detection ofoligonucleotides and nucleic acid hybridization techniques.Chemiluminescent reagents for HRP detection known in the art do notfully utilize the beneficial properties of this enzyme in analysismainly due to sensitivity limitations. More efficient chemiluminescentsubstrates are needed to improve the usefulness of this reporter enzyme.In addition, the ability to generate flashes of high intensity coupledwith an enzymatic amplification step, which is unknown in the art, couldprovide additional benefits in detection sensitivity and automateddetection.

a. Oxidation of Acridan

Applicants' U.S. Pat. No. 5,491,072 and co-pending applications, Ser.Nos. 08/205,093 filed Mar. 2, 1994 and 08/228,290 filed Apr. 15, 1994the disclosures of which are incorporated herein by reference, disclosethe use of a peroxidase enzyme to oxidize substituted and unsubstitutedN-alkylacridan-carboxylic acid derivatives to generatechemiluminescence. In the presence of a peroxidase enzyme and aperoxide, N-alkylacridancarboxylic acid derivatives are efficientlyoxidized to produce the N-alkylacridone and blue chemiluminescence in aone-step process.

Esters of 10-methylacridan-9-carboxylic acid undergo autoxidation toN-methylacridone in dipolar aprotic solvents under strongly basicconditions to produce chemiluminescence (F. McCapra, Acc. Chem. Res.,9(6), 201-8 (1976); F. McCapra, M. Roth, D. Hysert, K. A. Zaklika inChemiluminescence and Bioluminescence, Plenum Press, New York, 1973, pp.313-321; F. McCapra, Prog. Org. Chem., 8, 231-277 (1971); F. McCapra,Pure Appl. Chem., 24, 611-629 (1970); U.S. Pat. Nos. 5,283,334 and5,284,951 to McCapra and 5,284,952 to Ramakrishnan). Chemiluminescencequantum yields ranged from 10⁻⁵ to 0.1 and were found to increase as thepK_(a) of the phenol or alcohol leaving group decreased. Quantum yieldsin aqueous solution were significantly lower due a competingnon-luminescent decomposition of an intermediate. Addition of thecationic surfactant CTAB increased the apparent light yield 130-fold bypreventing a competing dark reaction.

b. Chemuliminescent Oxidation of Acridinium Esters

The chemiluminescent oxidation of aliphatic and aromatic esters ofN-alkylacridinium carboxylic acid by H₂ O₂ in alkaline solution is awell known reaction. The high chemiluminescence quantum yieldapproaching 0.1 has led to development of derivatives with pendantreactive groups for attachment to biological molecules. Numerouschemiluminescent immunoassays and oligonucleotide probe assays utilizingacridinium ester labels have been reported.

The use of acridinium esters (AE's), especially when labeled to aprotein or oligonucleotide suffers from two disadvantages. The chiefproblem is limited hydrolytic stability. Acridinium ester conjugatesdecompose steadily at or slightly above room temperature by hydrolysisof the ester group. Depending on the substitution of the leaving groupstorage at -20° C. may be required for extended storage. Amides,thioesters and sulfonimides of N-alkylacridinium carboxylic also emitlight when oxidized under these conditions (T. Kinkel, H. Lubbers, E.Schmidt, P. Molz, H. J. Skrzipczyk, J. Biolumin. Chemilumin., 4,136-139, (1989), G. Zomer, J. F. C. Stavenuiter, Anal. Chim. Acta, 227,11-19 (1989)). These modified leaving groups only partially improvestorage stability.

A second disadvantage of acridinium esters is the tendency to addnucleophiles such as water at the 9-position to form a non-luminescentpseudo-base intermediate which decomposes in a pH-dependent manner in adark process. In practice, the pH of solutions containing acridiniumesters must be first lowered to reverse pseudo-base formation and thenraised in the presence of H₂ O₂ to produce light.

A more fundamental limitation to the use of acridinium esters aschemiluminescent labels lies in the fact that when used as directlabels, only up to at most about 10 molecules can be attached to aprotein or oligonucleotide. Coupled with the quantum efficiency forproducing a photon (≦10%), an acridinium ester-labeled analyte cangenerate at most one photon of light. In contrast, enzyme-labeledanalytes detected by a chemiluminescent reaction can potentiallygenerate several orders of magnitude more light per analyte moleculedetected by virtue of the catalytic action of the enzyme.

An attempt to increase the number of acridinium ester moleculesassociated with an analyte in an immunoassay was made by constructing anantibody-liposome conjugate wherein the liposome contained anunspecified number of AE's (S.-J. Law, T. Miller, U. Piran, C. Klukas,S. Chang, J. Unger, J. Biolumin. Chemilumin., 4, 88-98, (1989)). Thismethod only produced a modest increase in signal over a comparable assayusing directly labeled AE's.

c. Chemiluminescent Detection of Horseradish Peroxidase

Amino-substituted cyclic acylhydrazides such as luminol and isoluminolreact with H₂ O₂ and a peroxidase enzyme catalyst (such as horseradishperoxidase, HRP) under basic conditions with emission of light. Thisreaction has been used as the basis for analytical methods for thedetection of H₂ O₂ and for the peroxidase enzyme. An analog of luminol(8-amino-5-chloro-7-phenylpyrido 3,4-d!pyridazine-1,4(2H,3H)dione) hasbeen used in an enhanced chemiluminescent assay with HRP (M. Ii, H.Yoshida, Y. Aramaki, H. Masuya, T. Hada, M. Terada, M. Hatanaka, Y.Ichimori, Biochem. Biophys. Res. Comm., 193(2), 540-5 (1993)). Anotherchemiluminescent compound oxidized by a peroxidase enzyme and a peroxideis a hydroxy-substituted phthalhydrazide (Akhavan-Tafti co-pending U.S.patent application No. 965,231, filed Oct. 23, 1992). Applicant'sco-pending application Ser. Nos. 08/061,810, 08/205,093 and 08/228,290disclose chemiluminescent N-alkylacridancarboxylic acid esters,thioesters and sulfonimides which produce light upon reaction with aperoxide and a peroxidase for use in detecting peroxidase enzymes and inassays.

Numerous enhancers have also been employed in conjunction with the useof luminol to increase the intensity and duration of light emitted.These include benzothiazole derivatives such as D-luciferin, variousphenolic compounds such as p-iodophenol and p-phenylphenol and aromaticamines (G. Thorpe, L. Kricka, in Bioluminescence and Chemiluminescence,New Perspectives, J. Scholmerich, et al, Eds., pp. 199-208 (1987)). Forthe purposes of the present-discussion phenolic compounds are taken tomean hydroxylic aromatic compounds which will also include compoundssuch as 2-naphthol and 6-bromo-2-naphthol which are known to enhanceother peroxidase reactions in addition to the aforementioned substitutedhydroxyphenyl compounds. Other compounds which function as enhancers ofthe chemiluminescent oxidation of amino-substituted cyclicacylhydrazides by a peroxidase are disclosed in U.S. Pat. No. 5,206,149to Oyama and No. 5,171,668 to Sugiyama, PCT application WO 93/16195dated Aug. 19, 1993 and in M. Ii, et al (infra).

OBJECTS

It is therefore an object of the present invention to provide animproved method and acridan compounds, especially arylN-alkylacridancarboxylate derivatives with superior properties for usein generating chemiluminescence by the action of a peroxidase enzyme forthe detection of biological materials and compounds. It is also anobject of the present invention to provide an improved method and kitusing aryl N-alkylacridancarboxylate derivatives for use in generatingchemiluminescence by the action of a peroxidase enzyme for the detectionof peroxidase enzymes and enzyme-conjugates in solution assays.Additionally, it is an object of the present invention to provide animproved method and kit using aryl N-alkylacridancarboxylate derivativesfor use in generating chemiluminescence by the action of a peroxidaseenzyme for use in nucleic acid assays in solution and on surfaces.Further, it is an object of the present invention to provide an improvedmethod and kit using aryl N-alkylacridancarboxylate derivatives for usein generating chemiluminescence by the action of a peroxidase enzyme fordetection of haptens, proteins and antibodies in enzyme immunoassays.

IN THE DRAWINGS

FIG. 1 is a graph showing the light emission profile from a reagentcontaining 2',3',6'-trifluorophenyl1,6-dimethoxy-10-methylacridan-9-carboxylate (5c) of the presentinvention. Forty μL of a formulation was incubated with 1 μL of asolution containing 1.4×10⁻¹⁶ mol of HRP in water. The formulationconsisted of: 1.5 μM acridan compound 5c in 0.01M tris buffer, pH 8.0,0.6 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 1 mM EDTA.After 100 sec, 100 μL of 0.1M NaOH was injected. The figure shows theintense burst of light emission (in Relative Light Units, RLU) generatedunder these conditions.

FIG. 2 is a graph showing the linearity of detection of HRP using areagent composition of the present invention. In separate experiments,50 μL of a solution containing acridan 5c were mixed at room temperaturewith 1.25 μL aliquots of HRP containing the indicated amounts of enzyme.After 100 sec, 100 μL of 0.1M NaOH was injected. Light intensity wasintegrated for 2 sec. The term S-B refers to the chemiluminescencesignal (S) in RLU in the presence of HRP corrected for backgroundchemiluminescence (B) in the absence of HRP.

FIG. 3 is a graph showing a series of absorption spectra from reactionof the reagent of Example 11 containing acridan 5c (3 mL) with 1.1×10⁻¹³mol of HRP. The absorbance spectrum was scanned from 300-500 nm at 30sec intervals after addition of enzyme. The progression of curves showsthe formation (in the direction bottom curve to top curve at 400 nm) ofthe acridinium compound 4c with an isosbestic point at about 338 nm.After 15 min, no further change was observed in the spectrum.

FIG. 4 is a graph showing a series of absorption spectra from reactionof 3 mL of a reagent containing the acridan compound2',6'-difluorophenyl 10-methylacridan-9-carboxylate with 1.1×10⁻¹³ molof HRP. The absorbance spectrum was scanned from 300-500 nm at 30 secintervals after addition of enzyme. The progression of curves shows amore complex behavior with no isosbestic point indicating the formation(in the direction bottom curve to top curve at 400 nm) of both theacridinium compound 2',6'-difluorophenyl10-methylacridinium-9-carboxylate and 10-methylacridone as proven bycomparison with authentic samples of these two compounds.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for producingchemiluminescence which comprises:

a) reacting a peroxide compound and a peroxidase enzyme with an acridanunder conditions of time, temperature and pH which permit theaccumulation of an intermediate compound wherein the acridan has theformula: ##STR1## wherein R is selected from alkyl, heteroalkyl andaralkyl groups, wherein R₁ to R₈ are selected independently from groupswhich allow the production of light and wherein Y is a leaving group;and

b) raising the pH to a level high enough to cause the production of aburst of light from reaction of the intermediate with peroxide at anintensity substantially greater than that being produced before raisingthe pH.

The present invention also relates to the use of this method fordetecting an analyte selected from haptens, antigens, proteins andantibodies, DNA, RNA and oligonucleotides in an assay procedure by achemiluminescent reaction, wherein the analyte is linked to or capableof being linked directly or indirectly to a peroxidase enzyme andwherein the amount of light produced is related to the amount of theanalyte.

The present invention also relates to the use of this method fordetecting a peroxidase enzyme in an assay procedure by achemiluminescent reaction, wherein the amount of light produced isrelated to the amount of enzyme. The enzyme may be free, in which caseit is the analyte, or linked to a member of a specific binding pair, forexample, by using a biotin-labeled analyte and streptavidin-peroxidaseconjugate. Other high affinity binding pairs well known in the art suchas fluorescein and anti-fluorescein, digoxigenin and anti-digoxigenin orcomplementary nucleic acid sequences may also be readily employed as ameans of linking a peroxidase enzyme to a member of a specific bindingpair for the purpose of practicing this invention. The method may thusbe used to detect haptens, antigens, proteins and antibodies by thetechnique of immunoassay and DNA or RNAby nucleic acid hybridizationassays.

The present invention also relates to the use of this method fordetecting hydrogen peroxide in an assay procedure by a chemiluminescentreaction with an acridan and a peroxidase enzyme, wherein the amount oflight produced is related to the amount of the peroxide present. It willbe apparent to those skilled in the art of chemiluminescent assays thatthe present methods can be used to detect oxidase enzymes anddehydrogenase enzymes. These enzymes can generate hydrogen peroxidethrough reduction of oxygen and oxidation of their native substrates.The hydrogen peroxide thereby produced can then be further reactedeither concurrently as it is generated or in a subsequent step with anacridan compound of the present invention and a peroxidase to producelight. A property of the light produced is then related to the amount ofthe oxidase or dehydrogenase enzyme. Further the oxidase ordehydrogenase enzyme may be present as a conjugate to a biologicalmolecule or a member of a specific binding pair in an assay for ananalyte.

The present invention also contemplates kits for detecting any of ananalyte, a peroxidase enzyme, a peroxidase enzyme conjugate, a peroxideor a reagent system or enzyme which produces hydrogen peroxide in anassay procedure by a chemiluminescent reaction. Kits useful forpracticing the present invention in any of its embodiments will comprisein one or more containers:

a) an acridan compound as described above;

b) a reagent for raising the pH of the reaction solution;

c) a peroxide if the analyte to be detected is not the peroxide or areagent which generates peroxide;

d) a peroxidase enzyme, if the analyte to be detected is not theperoxidase or a conjugate of a peroxidase with the analyte or aconjugate of a peroxidase with a reagent which forms a specific bindingpair with the analyte.

In another aspect the present invention relates to particular acridancompounds of the formula: ##STR2## wherein R is selected from alkyl,heteroalkyl and aralkyl groups, wherein R₁ to R₈ are selectedindependently from groups which allow the production of light andwherein at least one of R₁ and R₈ is a group selected from alkyl, alkoxyand halogen groups and wherein Y is a leaving group which allows theproduction of light from the acridan by reaction with a peroxide and aperoxidase. The leaving group Y can be any group which allows theproduction of light including, without limitation, aryloxy such asphenoxyand naphthyloxy, alkylthio and arylthio, sulfonimide and otherleaving groups known in the art.

Preferred groups of compounds are: ##STR3## wherein R is an alkyl,aralkyl or heteroalkyl group, wherein R₂₋₈ are selected independentlyfrom groups which allow the light to be produced, wherein OR₉ is a C₁ toC₂₀ straight or branched chain alkoxy group, wherein Ar is substitutedor unsubstituted aryl or heteroaryl and wherein R₁₀ is Ar or substitutedor unsubstituted alkyl, aralkyl or heteroalkyl.

Another class of preferred compounds is: ##STR4## wherein at least oneof R₂ through R₈ which may be the same or different are C₁ to C₂₀straight or branched chain alkoxy groups and wherein OR₉, R, R₁₀ and Arare as defined above.

Another class of preferred compounds is: ##STR5## wherein R is an alkyl,aralkyl or heteroalkyl group, wherein R₂₋₈ are selected independentlyfrom groups which allow the light to be produced, wherein R₁ is selectedfrom halogens and C₁ to C₂₀ straight or branched chain alkyl groups andwherein Ar is a substituted or unsubstituted aryl or heteroaryl group.

Acridan compounds useful in the practice of the present inventioninclude, without limitation, those with Ar or R₁₀ groups consisting ofsubstituted or unsubstituted aryl selected from phenyl, naphthyl,anthryl, phenanthryl and pyrenyl, heteroaryl selected from pyridyl,pyrimidinyl, pyridazinyl, quinolinyl, furyl, benzofuryl, thienyl,imidazolyl and the like. Groups which are contemplated as substituentsinclude alkyl, alkenyl, alkynyl, aralkyl, aryl, alkoxyl, alkoxy-alkyl,hydroxyalkyl, halogen, carbonyl, carboxyl, carboxamide, cyano,trifluoromethyl, amino, trialkylammonium and nitro groups.

Modifications of the particular combinations of the groups R, R₁₀ and Arcan be readily made in order to optimize the properties of the acridancompound for particular applications without departing from the scope ofthe present invention. For example, substituents may be selected forease of synthesis or to provide a compound with improved solubility orwith particular reaction kinetics. Substituents may also be chosen toprovide acridan compounds which have superior stability or diminish sidereactions or improve chemiluminescence efficiency as will be appreciatedby consideration of the reaction process detailed below and by referenceto the examples.

The present invention involves improved acridan compounds which, uponreaction with a peroxidase enzyme and a peroxide compound, are convertedinto an intermediate acridinium compound, wherein the center ring isaromatic, which subsequently undergoes a rapid chemiluminescent reactionat higher pH. Conducting the chemiluminescent reaction in this mannerresults in a brief burst of light with a high peak intensity. Incontrast, light generated by the method disclosed in U.S. Pat. No.5,491,072 rises gradually over several minutes to a steady level.Reaction of the acridan with the peroxidase and peroxide will normallybe carried out in an aqueous buffer solution at a pH which is compatiblewith enzyme activity, preferably between about 6 and about 8.5. The pHof the solution is then increased to above about 11 after a preliminaryincubation period of a few seconds to several minutes. The intermediateformed by the enzymatic reaction produces a burst of luminescence byreaction with peroxide at the higher pH. ##STR6##

The rate of the chemiluminescent decomposition of the acridiniumcompound during the enzymatic oxidation phase can be slowed byappropriate choice of acridan compound or by adjusting reactionconditions allowing the acridinium compound to accumulate. The rate ofautoxidation of the unreacted acridan compound can also be slowed byappropriate choice of ring substituents. For example, acridans withsubstituents other than hydrogen at the 1-position and acridans with twoor more substituents selected from alkoxy, alkyl and halogen exhibitlower background (non-enzymatic) chemiluminescence and are oxidized toacridinium compounds with better stability. Subsequently making thereaction solution highly basic greatly accelerates the reaction of theacridinium with peroxide to expel the leaving group and CO₂ and producelight arising from the excited state of the N-substituted acridone.

Acridinium ester, thioester or sulfonimide compounds of the formula:##STR7## which are formed by the peroxidase-catalyzed oxidation of thecorresponding acridan compound according to the present reaction areunexpectedly stable to peroxide under slightly acidic, neutral ormoderately alkaline pH. Since the acridinium compound is formed in thepresence of excess peroxide, any significant reactivity of theacridinium compound with the excess peroxide would lead to continuouslight emission as the enzymatic reaction proceeds. Acridan compounds ofthe present invention produce acridinium compounds which are relativelyunreactive to peroxide under the reaction conditions.

The chemiluminescent reaction of the present invention provides anunexpectedly sensitive method for detection of peroxidase enzymes orperoxide compounds. The analytical sensitivity as defined by thesignal/background ratio is limited by the ability to distinguish thelight produced by the base-induced reaction of the enzymaticallyproduced intermediate from all other light producing processes. Quiteunexpectedly, three potentially problematic side reactions do not takeplace to an extent that interferes with the measurement of the desiredsignal. First, the acridinium ester intermediates formed by enzymaticoxidation of the acridan in the present method produce relatively lowlevels of light at neutral to moderately alkaline pH. This is surprisingin view of the fact that acridinium esters, thioesters and sulfonimidesknown in the art react rapidly with hydrogen peroxide to produce intensechemiluminescence.

Second, N-alkylacridancarboxylate esters themselves undergo achemiluminescent reaction (autoxidation) with molecular oxygen at pH≧11as discussed in McCapra, infra. Any unreacted acridan remaining afterthe incubation with enzyme would therefore be expected to produce alarge background emission when the pH was raised. Surprisingly, theacridans of the present invention do not generate a significant quantityof light at the high pH used in the second step relative to the level oflight produced from the enzymatically generated acridinium compound.

Third, acridinium compounds can undergo side reactions which do notproduce light. Reactions well known in the art which consume theacridinium compound by competing non-luminescent pathways will decreasethe amount of light which can be produced. Hydrolysis results inexpulsion of the leaving group Y and formation of a non-luminescentcarboxylate ion. Addition of nucleophiles to the 9-position results inan intermediate termed a pseudo-base. While this reaction is reversibleby lowering the solution pH to about 1 to 3, this would unnecessarilycomplicate the reaction. Hydrolysis of the starting acridan as wellwould limit the amount of light which could be produced.

The reaction of the present invention is carried out in solution such asan aqueous buffer which may be in contact with the surface of a solidsupport such as a bead, tube, membrane or microwell plate coated withenzyme. Suitable buffers include any of the commonly used bufferscapable of maintaining a pH in the range of about 6 to about 8.5 forexample, phosphate, borate, carbonate, tris(hydroxymethylamino)methane,glycine, tricine, 2-amino-2-methyl-1-propanol, diethanolamine and thelike. The preferred method of practicing the invention in this regard isdetermined by the requirements of the particular intended use.

Incorporation of certain enhancer compounds either alone or incombination with surfactants into the reaction mixture promotes thereactivity of the enzyme. Since the enzymatically produced intermediateundergoes a subsequent chemiluminescent reaction upon raising the pH,the enhanced production of intermediate translates to enhancedproduction of light. Included among these enhancers are phenoliccompounds and aromatic amines known to enhance other peroxidasereactions as described in G. Thorpe, L. Kricka, in Bioluminescence andChemiluminescence, New Perspectives, J. Scholmerich, et al, Eds., pp.199-208 (1987), M. Ii, H. Yoshida, Y. Aramaki, H. Masuya, T. Hada, M.Terada, M. Hatanaka, Y. Ichimori, Biochem. Biophys. Res. Comm., 193(2),540-5 (1993), and in U.S. Pat. Nos. 5,171,668 and 5,206,149 which areincorporated herein by reference. Substituted and unsubstitutedarylboronic acid compounds and their ester and anhydride derivatives asdisclosed in PCT WO 93/16195, Aug. 19, 1993 and incorporated herein byreference are also considered to be within the scope of enhancers usefulin the present invention. Preferred enhancers include but are notlimited to: p-phenylphenol, p-iodophenol, p-bromophenol,p-hydroxy-cinnamic acid, 2-naphthol and 6-bromo-2-naphthol.

Additives which suppress the generation of chemiluminescence from thereaction of hydrogen peroxide and aryl acridan derivatives in theabsence of peroxidase enzymes are employed to further improve theutility of the invention. It has also been found that certainsurfactants such as anionic, cationic and nonionic surfactants improvethe sensitivity of detection of the peroxidase enzyme in assays of thepresent invention by providing a larger signal.

The preferred amounts of the various components of a composition of thepresent invention are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Acridan              1 nM-1 mM                                                Phenol enhancer      1 μm-10 mM                                            Surfactant           0.005-5%                                                 Peroxide             0.01-10 mM                                               Chelating agent      0.01-5 mM                                                ______________________________________                                    

The present invention involves a solution in an aqueous buffercontaining 1) a phenol enhancer or a salt of a phenol enhancer, 2) aperoxide compound wherein the peroxide compound may be, withoutlimitation, hydrogen peroxide, urea peroxide, or a perborate salt, 3) anacridan compound of the invention, 4) a polydentate cation complexingagent such as EDTA, EGTA and their salts, and 5) a surfactant such asthe anionic surfactant sodium dodecyl sulfate (SDS), or preferably anonionic surfactant such as polyoxyethylenated alkylphenols,polyoxyethylenated alcohols, polyoxyethylenated ethers,polyoxyethylenated sorbitol esters and the like.

In a preferred method of practicing the present invention, an aqueousbuffer solution with a pH in the range of about 5 to about 9 containinga phenol compound such as p-phenylphenol or p-iodophenol at a finalconcentration from about 0.01M to 1×10⁻⁶ M, a nonionic surfactant at afinal concentration from about 5% to 0.005% (v/v), a peroxide sourcesuch as hydrogen peroxide or, preferably, a perborate salt or ureaperoxide and a cation complexing agent such as EDTA at a finalconcentration from about 1×10⁻³ M to 1×10⁻⁵ M is mixed with a secondsolution containing an acridan compound of the invention to achieve afinal acridan concentration from about 0.001M to about 1×10⁻⁹ M to formthe detection reagent solution. This solution is contacted with theperoxidase enzyme which may either be in solution or adhered to a solidsupport. The detection reaction may be performed over a range oftemperatures including at least the range 10°-40° C. After an incubationperiod, the pH of the solution is raised to at least about 10 byaddition of a base and, optionally, additional peroxide. As a result,light is produced which rapidly rises to a maximum level and decays.Preferably the addition of base is done rapidly so that the light isemitted over a time interval of a few seconds. Adjustments of incubationtime and temperatures and reaction pH as are apparent to the skilledartisan are considered to be within the subject matter of the invention.

A significant advantage of aryl acridan derivatives and compositions ofthe present invention containing them includes the ability to measureall of the light emitted from the accumulated chemiluminescent productin a short period of time. Measurement of the total light emission whichoccurs within a period of a few seconds is equivalent to integrating theintensity vs. time curve produced by reaction of applicant's previouslydisclosed acridans which produce an extended emission. As a result ofthe time compression of light emission, very small amounts of peroxidaseenzyme activity yield large, easily measured spikes of light. This canlead to improved sensitivity of detection if backgroundchemiluminescence is controlled. Assays designed with this type of lightdetection are readily adapted to existing high volume commercialimmunoassay instruments. These and other advantages will be apparent byconsideration of the examples.

EXAMPLES Synthesis of Acridan Derivatives

Acridancarboxylic acid derivatives 5a-h were synthesized according toone of the methods shown in Scheme 2 from the correspondingacridine-9-carboxylic acid. In the structure shown below, the identityand position of substituents A and B are explained in the table. Allother substituents are H.

                  TABLE II                                                        ______________________________________                                         ##STR8##                                                                     Compound A        B        X    Ar                                            ______________________________________                                        5a       3-OCH.sub.3                                                                            H        O    2',3',6'-Trifluorophenyl                               4-Cl                                                                 5b       3-OCH.sub.3                                                                            H        S    4'-Fluorophenyl                                        4-Cl                                                                 5c       1-OCH.sub.3                                                                            6-OCH.sub.3                                                                            O    2',3',6'-Trifluorophenyl                      5d       OCH.sub.3                                                                              OCH.sub.3                                                                              O    2',3',6'-Trifluorophenyl                               Cl       Cl                                                          5e       3-OCH.sub.3                                                                            6-OCH.sub.3                                                                            O    2',3',6'-Trifluorophenyl                      5g       1-CH.sub.3                                                                             H        O    2',3',6'-Trifluorophenyl                      5h       1-Cl     H        O    2',3',6'-Trifluorophenyl                      5i       1-OCH.sub.3                                                                            6-OCH.sub.3                                                                            O    2',3',6'-Trifluorophenyl                               4-CH.sub.3                                                           5j       1-CH.sub.3                                                                             H        O    2',3',6'-Trifluorophenyl                               4-CH.sub.3                                                           5k       1-OCH.sub.3                                                                            H        O    2',3',6'-Trifluorophenyl                               4-OCH.sub.3                                                          ______________________________________                                    

The structure of compound 5d has not been completely determined sincethe position of the OCH₃ and Cl substituents is not known withcertainty. The simplicity of the ¹ H NMR spectrum indicates a highdegree of symmetry. ##STR9## The acridine-9-carboxylic acid compounds1c, e, f, g and h were prepared by literature methods (G. Zomer, J.Stavenuiter, R. Van Den Berg, E. Jansen, In Luminescence Techniques inChemical and Biochemical Analysis, W. Baeyens, D. De Keukeleire, K.Korkidis, eds., Dekker, New York, 505-521, (1991); R. Stolle, J. Prakt.Chem., 105, 137, (1922)). The acid chloride 2a (A=3--OCH₃, 4-Cl; B=H)was produced during the reaction of 1f with SOCl₂ in addition to 2f.

                  TABLE III                                                       ______________________________________                                         ##STR10##                                                                    Compound      A            B                                                  ______________________________________                                        1c            1-OCH.sub.3  6-OCH.sub.3                                        1e            3-OCH.sub.3  6-OCH.sub.3                                        1f            3-OCH.sub.3  H                                                  1g            1-CH.sub.3   H                                                  1h            1-Cl         H                                                  1i            1-OCH.sub.3  4-CH.sub.3                                                                    6-OCH.sub.3                                        1j            1-CH.sub.3  4-CH.sub.3                                                                     H                                                  1k            1-OCH.sub.3  4-OCH.sub.3                                                                   H                                                  ______________________________________                                    

Example 1 Synthesis of Compound 5a

2',3',6'-Trifluorophenyl4-chloro-3-methoxy-10-methylacridan-9-carboxylate.

(a) Condensation of the commercially available (Aldrich)3-methoxydiphenylamine with oxalyl chloride produced3-methoxyacridinecarboxylic acid (1f) which was used to prepare acidchloride 2a.

(b) Compound 1f was converted to a mixture of compounds 2a and 2f (1.5g) by refluxing in 10 mL of SOCl₂ for 3 h. The solvent was removed underreduced pressure to obtain a yellow solid which was dissolved inmethylene chloride (CH₂ Cl₂) and pyridine (0.7 mL) under argon. Asolution of the phenol (0.878 g) in CH₂ Cl₂ was added dropwise. Thesolution was stirred overnight at room temperature then diluted withmore CH₂ Cl₂ (100 mL) and washed with water (3×50 mL). The organic layerwas dried over Na₂ SO₄ and concentrated to obtain a mixture of esters 3aand 3f. The product 2',3',6'-trifluorophenyl 4-chloro-3-methoxyacridine-9-carboxylate (3a) was isolated by chromatography onsilica with 25% ethyl acetate/hexane: ¹ H NMR (CDCl₃) δ 4.15 (s, 3H),7.1 (m, 1H), 7.2 (m, 1H), 7.59 (d, 1H), 7.65 (ddd, 1H), 7.85 (ddd, 1H),8.17 (dt, 2H), 8.39 (dt, 1H); ¹³ C NMR (CDCl₃) δ 57, 110.0, 114.7,116.7, 117.2, 118.9, 121.3, 124.5, 124.6, 127.6, 127.8, 130.3, 131.2,134.9, 144.2, 145.5, 148.5, 149.3, 150.0, 156.0, 163.7. The structure ofthis compound was verified by ¹ H-¹³ C NOE experiments, a ¹ H-coupled ¹³C NMR spectrum and a ¹ H-¹³ C 2D NMR spectrum which established theposition of substitution of the OCH₃ and Cl groups and massspectrometry. MS (m/z) 417, 270 (100), 242, 164.

(c) Ester 3a (0.223 g) was dissolved in 70 mL of ethanol with heatingunder an atmosphere of argon. After cooling to room temperature andshielding the flask from light, 0.313 g of NH₄ Cl (10 eq. ) was addedfollowed by 0.382 g (10 eq.) of zinc dust. After 90 min, the solids werefiltered off, washed with CH₂ Cl₂ and the combined solvents evaporated.The resulting solid material was dissolved in CH₂ Cl₂ and filteredyielding a light yellow material identified as 2',3',6'-trifluorophenyl4-chloro-3 -methoxyacridan-9-carboxylate (6a) by TLC which showed thematerial to be pure and have an R_(f) different from the starting ester.

(d) The reduced product was methylated with methyl triflate (7 mL) inca. 8 mL of CH₂ Cl₂ under argon with protection from light. After 4 daysthe volatiles were evaporated and the product chromatographicallypurified on silica using 50% ethyl acetate/hexane yielding compound 5aas a white solid.

Example 2 Synthesis of Compound 5b

4'-Fluorophenyl 4-chloro-3-methoxy-10-methylacridan-9-thiocarboxylate.

(a) Acid 1f was converted to the acid chlorides 2a and 2f as describedin Example 1. The mixture of products 2a and 2f was dissolved in ca. 200mL of CH₂ Cl₂ under argon. Pyridine (4.2 mL, 1.3 eq.) was added and thesolution stirred for 15 min. 4-Fluorothiophenol (5.4 mL, 1.2 eq.) wasadded and the warm solution stirred over night. Additional CH₂ Cl₂ (100mL) was added and the solution extracted with water and saturated NaCl.The organic layer was dried over Na₂ SO₄ and concentrated to a brownsolid. The product 4'-fluorophenyl4-chloro-3-methoxyacridine-9-thiocarboxylate (3b) was isolated bychromatography on silica with solvents of graded polarity ranging from20% ethyl acetate/hexane to pure ethyl acetate: ¹ H NMR (CDCl₃) δ 4.162(s, 3H), 7.18-7.24 (m, 2H), 7.55-7.65 (m, 4H), 7.82-7.88 (m, 1H),8.07-8.10 (d, 2H), 8.35-8.38 (d, 1H).

(b) Thioester 3b (1.19 g) was dissolved in 300 mL of ethanol withheating. After cooling to room temperature and shielding the flask fromlight, 1.75 g of NH₄ Cl (10 eq.) was added followed by 2.14 g (10 eq.)of zinc dust. After 15 min, the solids were filtered off, washed withCH₂ Cl₂ and the combined solvents evaporated. The resulting solidmaterial was dissolved in CH₂ Cl₂ and filtered yielding a light brownmaterial identified as 4'-fluorophenyl1-methoxyacridan-9-thiocarboxylate (6b) by ¹ H NMR which was used withno additional purification for preparation of compound 5b: ¹ H NMR(CDCl₃) δ 3.928 (s, 3H), 5.165 (s, 1H), 6.56-6.59 (d, 1H), 6.89-7.03 (m,5H), 7.16-7.34 (m, 4H).

(c) The reduced product was methylated with methyl triflate (12 mL) in 5mL of CH₂ Cl₂ under argon with protection from light. After 72 hours thevolatiles were evaporated and the product chromatographically purifiedon silica using 10-20% ethyl acetate/hexane yielding 0.75 g of 5b as awhite solid: ¹ H NMR (CDCl₃) δ 3.712 (s) , 3.916 (s, 3H), 4.892 (s, 1H),6.66-6.69 (d, 1H), 6.97-7.06 (m, 3H), 7.12-7.27 (m, 5H), 7.31-7.37 (m,1H).

Example 3 Synthesis of Compound 5c

2',3',6'-Trifluorophenyl 1,6-dimethoxy-10-methylacridan-9-carboxylate.

(a) Reaction of bis(3-methoxyphenyl)amine (Aldrich) with AlCl₃ andoxalyl chloride followed by base-catalyzed rearrangement of the isatinsproduced a mixture of carboxylic acids. The mixed acids (1.4 g, 4.9mmol) were suspended in excess SOCl₂ (15 mL) and the reaction mixturewas refluxed for 4 h. The solvent was removed under reduced pressure andthe acid chloride product was combined with 2,3,6-trifluorophenol (0.74g, 5 mmol) and CH₂ Cl₂. Pyridine (1 mL, 13 mmol) was added dropwiseunder argon. The solution was stirred overnight at room temperature andthe volatiles removed under reduced pressure. The crude product wassubjected to column chromatography on silica using 10% ethylacetate/hexane. Pure 2',3',6'-trifluoro-phenyl1,6-dimethoxyacridine-9-carboxylate (3c) was thereby isolated from themixture of ester products along with two other esters designated 3d,whose structure is not precisely known, and 3e. Compound 3c: ¹ H NMR(CDCl₃) δ 4.03 (s, 3H), 4.04 (s, 3H), 6.85-6.88 (d, 1H), 7.02-7.24 (m,2H), 7.32-7.36 (dd, 1H), 7.48-7.49 (d, 1H), 7.70-7.83 (m, 2H), 8.08-8.11(d, 1H).

(b) Ester (3c) (0.2 g, 0.46 mmol) was methylated by overnight stirringin 10 mL of CH₂ Cl₂ with methyl trifluoromethanesulfonate (1 mL, 8.8mmol) under argon. Volatiles were evaporated under reduced pressure andthe residue washed with ethyl acetate. N-Methylacridinium ester (4c) wasused directly in the next step.

(c) Reduction to the acridan (5c) was accomplished by reacting asolution of 4c (45 mg) and 1 g of NH₄ Cl in 25 mL of ethanol with 1 g ofzinc. The yellow solution decolorized immediately and was stirred anadditional 30 min. Ethyl acetate (50 mL) was added and the mixturefiltered. Evaporation of solvents and chromatography of the residue onsilica with 10% ethyl acetate/hexane produced pure 5c: ¹ H NMR (CDCl₃) δ3.39 (s, 3H), 3.84 (S, 3H), 3.90 (S, 3H), 5.90 (s, 1H), 6.50-7.43 (m,8H).

Example 4 Synthesis of Compound 5d

2',3',6'-Trifluorophenyldichlorodimethoxy-10-methylacridan-9-carboxylate.

(a) Ester 3d was isolatedby chromatography from the experiment describedin Example 3. ¹ H NMR (CDCl₃) δ 4.17 (s, 6H), 7.10-7.30 (m, 2H),7.58-7.61 (d, 2H), 8.16-8.19 (d, 2H). Substitution of two chlorine atomson the acridine ring was shown by mass spectrometry. MS (m/z) 481, 334(100), 306. The substitution pattern of this product has not beendetermined unambiguously although the simplicity of the 1H NMR spectrumindicates that the compound is symmetrical.

(b) To a warm solution of ester (0.02 g) and ammonium chloride (2 g) inethanol (25 ml) was added zinc (2 g) causing immediate decolorization ofthe solution. The colorless solution was stirred at room temperature for30 min. Ethyl acetate (200 mL) was added to the solution which was thenfiltered. The solvents were removed from the filtrate under reducedpressure. The crude material obtained was chromatographed on silica gel(10% ethyl acetate/hexane) to yield the pure product (6d). ¹ H NMR(acetone-d₆) δ 3.99 (s, 6H), 5.66 (s, 1H), 6.87-6.89 (d, 2H), 7.12-7.40(m, 2H), 7.43-7.46 (d, 2H), 7.58 (s, 1H).

(c) The reduced product (0.16 g, 0.46 mmol) was methylated with methyltriflate (3 mL) in 5 mL of CH₂ Cl₂ under argon with protection fromlight. After 72 hours the volatiles were evaporated and the productchromatographically purified on silica using 30% ethyl acetate/hexaneyielding 5d as a white solid: ¹ H NMR (CDCl₃) δ 3.47 (s, 3H), 3.91 (s,3H), 5.03 (s, 1H), 6.74-6.77 (d, 2H), 7.80-7.00 (m, 2H), 7.21-7.23 (d,2H). As indicated above for 3d, the substitution pattern of this producthas not been determined unambiguously although the simplicity of the ¹ HNMR spectrum indicates that the compound is symmetrical.

Example 5 Synthesis of Compound 5e

2',3',6'-Trifluorophenyl 3,6-dimethoxy-10-methylacridan-9-carboxylate.

(a) Ester 3e was isolated by chromatography from the experimentdescribed in Example 3. ¹ H NMR (CDCl₃) δ 4.03 (s, 6H), 7.11-7.24 (m,2H), 7.29-7.33 (dd, 2H), 7.47-7.48 (d, 2H), 8.08-8.11 (d, 2H).

(b) Ester 3e (1 g, 2.3 retool) was methylated by overnight stirring in30 mL of CH₂ Cl₂ with methyl triflate (1.3 mL, 5 eq.) under argon.Volatiles were evaporated under reduced pressure and the residue washedwith ethyl acetate. N-Methylacridinium ester (4e) was used directly inthe next step. ¹ H NMR (DMSO-d₆) δ 4.25 (s, 6H), 4.73 (s, 3H), 7.60-8.23(m, 8H).

(c) Reduction to acridan 5e was accomplished by reacting a solution of4e (650 mg) and 650 mg of NH₄ Cl in 100 mL of ethanol with 650 mg ofzinc. The yellow solution decolorized immediately and was stirred anadditional 30 min. Ethyl acetate (150 mL) was added and the mixturefiltered. Evaporation of solvents and chromatography of the residue onsilica with 10% ethyl acetate/hexane produces pure 5e. ¹ H NMR(acetone-d₆) δ 3.48 (s, 3H), 3.88 (s, 6H), 5.41 (s, 1H), 6.62-7.39 (m,8H).

Example 6 Synthesis of Compound 5g

2',3',6'-Trifluorophenyl 1,10-dimethylacridan-9-carboxylate.

(a) Reaction of 3-methyldiphenylamine (Aldrich) with AlCl₃ and oxalylchloride followed by base-catalyzed rearrangement of the isatinsproduced a mixture of 1-methylacridine-9-carboxylic acid and3-methylacridine-9-carboxylic acid. The mixed acids (4 g, 15.6 mmol)were suspended in excess SOCl₂ (50 mL) and the reaction mixture wasrefluxed for 1.5 h. The solvent was removed under reduced pressure. Tothe above residue was added 2,3,6-trifluorophenol (2.77 g, 18.7 mmol).This mixture was dissolved in CH₂ Cl₂ and pyridine (4 ml, 49.5 mmol) wasadded dropwise under argon. The solution was stirred for several days atroom temperature, then the solvent and excess pyridine were removedunder reduced pressure. The crude material obtained was chromatographedon silica gel (5% ethyl acetate/hexane) to yield 1-methylacridan ester 3g and the isomeric 3-methyl ester. Compound 3 g: ¹ H NMR (CDCl₃) δ7.04-7.25 (m, 2H), 7.70-7.77 (m, 3H), 7.87-7.92 (t, 1H), 8.24-8.31 (m,3H).

(b) Ester 3g (0.2 g) and ammonium chloride (2 g) in ethanol (200 ml) aretreated with zinc (2 g) causing immediate decolorization of thesolution. The colorless solution is stirred at room temperature for 30min. Ethyl acetate (200 mL) is added to the solution which is thenfiltered. The solvents are removed from the filtrate under reducedpressure. The crude material is chromatographed on silica gel (40% ethylacetate/hexane) to yield 2',3',6'-trifluorophenyl1-methylacridan-9-carboxylate (6 g).

(c) The reduced product (0.18 g) is methylated with methyl triflate (3mL) in 5 mL of CH₂ Cl₂ under argon with protection from light. Afterabout 72 hours the volatiles are evaporated and the productchromatographically purified on silica using 30% ethyl acetate/hexane toyield 5 g as a white solid.

Example 7 Synthesis of Compound 5h

2',3',6'-Trifluorophenyl 1-chloro-10-methylacridan-9-carboxylate.

(a) Reaction of 3-chlorodiphenylamine (Aldrich) with AlCl₃ and oxalylchloride followed by base-catalyzed rearrangement of the isatinsproduced a mixture of 1-chloroacridine-9-carboxylic acid and3-chloroacridine-9-carboxylic acid. The mixed acids (4.0 g, 15.6 mmol)were suspended in excess SOCl₂ (50 mL) and the reaction mixture wasrefluxed for 1.5 h. The solvent was removed under reduced pressure. Tothe above residue was added 2,3,6-trifluorophenol (2.77 g, 18.7 mmol).This mixture was dissolved in CH₂ Cl₂ and pyridine (4 ml, 49.5 mmol) wasadded dropwise under argon. The solution was stirred for several days atroom temperature, then the solvent and excess pyridine were removedunder reduced pressure. The crude material obtained was chromatographedon silica gel (5% ethyl acetate/hexane) to yield 1-chloroacridine ester3 h and the isomeric 3-chloro ester.

Compound 3h: ¹ H NMR (CDCl₃) δ 7.04-7.25 (m, 2H), 7.70-7.77 (m, 3H),7.87-7.92 (m, 1H), 8.24-8.31 (m, 3H).

(b) To a warm solution of ester 3 h (0.2 g, 0.52 mmol) and ammoniumchloride (2 g) in ethanol (200 ml) was added zinc (2 g) causingimmediate decolorization of the solution. The colorless solution wasstirred at room temperature for 30 min. Ethyl acetate (200 mL) was addedto the solution which was then filtered. The solvents were removed fromthe filtrate under reduced pressure. The crude material obtained waschromatographed on silica gel (40% ethyl acetate/hexane) to yield thepure product 2,3,6-trifluorophenyl 1-chloroacridan-9-carboxylate (6 h).¹ H NMR (CDCl₃) δ 5.72 (s), 6.29 (s), 6.68-6.70 (d), 6.77-6.79 (d),6.79-6.88 (m), 6.96-7.03 (m), 7.12-7.17 (t), 7.21-7.27 (d), 7.54-7.76(d).

(c) The reduced product 6 h (0.18 g, 0.46 mmol) was methylated withmethyl triflate (3 mL) in 5 mL of CH₂ Cl₂ under argon with protectionfrom light. After 72 hours the volatiles were evaporated and the productchromatographically purified on silica using 30% ethyl acetate/hexaneyielding 5 h as a white solid: ¹ H NMR (CDCl₃) δ 3.441 (S, 3H), 5.754(s, 1H), 6.81-7.09 (m, 6H), 7.22-7.38 (m, 2H), 7.52-7.55 (dd, 1H).

Example 8 Synthesis of Compound 5i

2',3',6'-Trifluorophenyl1,6-dimethoxy-4,10-dimethylacridan-9-carboxylate.

(a) 5-Methoxy-2-methylaniline (9.77 g, Aldrich) was converted to theacetamide derivative by reaction with 8.7 mL of acetic anhydride, 8.2 mLof acetic acid and 43 mg of zinc at reflux for 7 h. The reaction mixturewas poured into 250 mL of ice water and stirred. The light brown solidwas filtered and air-dried yielding 6.37 g of the product: ¹ H NMR(CDCl₃) δ 1.579 (s,3H), 2.196 (s,3H), 3.791 (s,3H), 6.62-6.66 (dd, 1H),6.92 (m, 1H), 7.06-7.08 (d, 1H), 7.554 (s, 1H).

(b) 5-Methoxy-2-methylacetanilide (6.37 g) was condensed with3-bromoanisole (11.1 mL) in the presence of 5.06 g of K₂ CO₃ and 0.74 gof CuI at reflux for 8 h. After standing overnight, the mixture washeated and then extracted with toluene (3×100 mL) and evaporated to abrown oil.

The oil was dissolved in 150 mL of ethanol, 4 g of KOH were added andthe mixture refluxed for 9 h. The ethanol was evaporated and thered-brown solid taken up in 300 mL of water and extracted with ethylacetate. The ethyl acetate was evaporated and the crude solid partiallypurified by passing a CH₂ Cl₂ solution through a plug of silica. Finalpurification was effected by column chromatography on silica (5% ethylacetate/hexane) to produce 7.63 g of the diphenylamine compound: ¹ H NMR(CDCl₃) δ 2.194 (s,3H), 3.752 (s, 3H), 3.783 (s, 3H), 5.404 (s, 1H),6.47-6.52 (m, 2H), 6.55-6.62 (m, 2H), 6.85-6.86 (d, 1H), 7.08-7.10 (d,1H), 7.14-7.20 (t, 1H).

(c) Reaction of 5-methoxy-2-methylphenyl-3'-methoxyphenylamine (7.63 g)with 3.12 mL of oxalyl chloride in 110 mL of CH₂ Cl₂ and followed byreaction with 8.36 g of AlCl₃ produced the isatin which was converted bybase-catalyzed rearrangement with 100 mL of 10% KOH and neutralizationto 1,6-dimethoxy-4-methylacridine-9-carboxylic acid (1i), (8.75 g): ¹ HNMR (CD₃ OH/KOH) δ 2.168 (s, 3H), 3.706 (s, 3H), 3.763 (s, 3H),6.30-6.34 (m, 2H), 6.70-6.74 (dd, 1H), 6.88-6.89 (d, 1H), 7.184-7.213(d, 1H), 7.693-7.721 (d, 1H).

(d) Acid 1i (1.0 g) was dissolved in pyridine (20 mL). p-Toluenesulfonylchloride (1.28 g) was added and the reaction mixture was stirred for 1h. 2,3,6-Trifluorophenol (1.0 g) in 5 mL of pyridine was added and thesolution was stirred overnight at room temperature. The ester productwas isolated by chromatography on silica gel (20-40% ethylacetate/hexane). A second chromatogratphic purification using 50% CH₂Cl₂ /hexane yielded 110 mg of ester 3i: ¹ H NMR (CDCl₃) δ 2.841 (s, 3H),4.003 (s, 3H), 4.051 (s, 3H), 6.75-6.78 (d, 1H), 7.04-7.23 (m, 2H),7.31-7.36 (dd, 1H), 7.53-7.57 (m, 2H), 8.08-8.11 (d, 1H). Attemptedpreparation of the ester by making the acid chloride with SOCl₂ resultedin chlorination of the acridine ring.

(e) To a slurry of ester 3i (110 mg) and NH₄ Cl (138 mg) in Ar-purged2-propanol (25 mL) was added zinc (168 mg) causing immediatedecolorization of the solution. The colorless solution was stirred atroom temperature for 2.5 h shielded from light. CH₂ Cl₂ was added to thesolution which was then filtered. The solvents were removed from thefiltrate under reduced pressure. The crude material was purified byprep. TLC (20% ethyl acetate/hexane) to yield 29.5 mg of acridan 6i: ¹ HNMR (CDCl₃) δ 2.204 (s, 3H), 3.799 (s, 3H), 3.850 (s, 3H), 5.534 (s,1H), 6.025 (s, 1H), 6.31-6.32 (d, 1H), 6.35-6.39 (d, 1H), 6.50-6.54 (dd,1H), 6.80-7.03 (m, 3H), 7.42-7.45 (d 1H).

(f) The reduced product, acridan 6i (29.5 mg) was methylated with methyltrillate (0.5 mL) in 6 mL of CH₂ Cl₂ under argon with protection fromlight. After 48 h, another 1 mL of methyl triflate was added andstirring continued for another day. The volatiles were evaporated andthe product purified by prep. TLC using 20% ethyl acetate/hexaneyielding 5i as a white solid: ¹ H NMR (CDCl₃) δ 2.373 (s, 3H), 3.478 (s,3H), 3.838 (s, 3H), 3.876 (s, 3H), 5.447 (s, 1H), 6.53-6.64 (m, 3H),6.77-7.01 (m, 2H), 7.07-7.10 (d, 1H), 7.30-7.34 (d, 1H).

Example 9 Synthesis of Compound 5i

2',3',6'-Trifluorophenyl 1,4,10-trimethylacridan-9-carboxylate.

(a) 2,5-Dimethylaniline (30 g, Aldrich) was converted to the acetamidederivative by reaction with 30.4 mL of acetic anhydride, 28.3 mL ofacetic acid and 140 mg of zinc at reflux for 3 h. The reaction mixturewas poured into 700 mL of ice water and stirred. The white solid wasfiltered, washed with water and air-dried yielding 24.88 g of theproduct: ¹ H NMR (CDCl₃) δ 1.561 (s, 3H), 2.221 (s, 3H), 2.322 (S, 3H),6.88-6.92 (d, 1H) 7.05-7.09 (d, 1H), 7.61 (s, 1H).

(b) 2,5-Dimethylacetanilide (12.75 g) was condensed with 3-bromoanisole(25 mL) in the presence of 11.12 g of K₂ CO₃ and 1.62 g of CuI at refluxfor 4 d. After standing overnight, the mixture was heated and extractedwith toluene (3×100 mL) and evaporated to a brown oil/solid mixture.

The oil was dissolved in 300 mL of ethanol, 8.77 g of KOH were added andthe mixture refluxed for 24 h. The ethanol was evaporated and the oiltaken up in 300 mL of water and extracted with CH₂ Cl₂ (3×175 mL). Thecombined CH₂ Cl₂ extracts were washed with water and purified by passingthe solution through a plug of Na₂ SO₄ /silica/Na₂ SO₄ which produced10.25 g of the diphenylamine compound: ¹ H NMR (CDCl₃) δ 2.225 (s, 3H),2.288 (s, 3H), 5.353 (s, 1H), 6.76-6.78 (d, 1H), 6.89-6.98 (m, 3H),7.08-7.11 (d, 2H), 7.24-7.29 (t, 2H).

(c) Reaction of 2,5-dimethyldiphenylamine (10.25 g) with 5.21 mL ofoxalyl chloride in 70 mL of CH₂ Cl₂ at reflux for 45 min. followed byreaction with 13.86 g of AlCl₃ produced the isatin which was convertedto 1,4-dimethylacridine-9-carboxylic acid (1j) (9.88 g) bybase-catalyzed rearrangement with 125 mL of 10% KOH at reflux andneutralization with 500 mL of a 3:1 ice/5M HCl mixture: ¹ H NMR(DMSO-d₆) δ 2.80-2.82 (d, 6H), 7.40-7.42 (d, 1H), 7.63-7.66 (d, 1H),7.70-7.75 (t, 1H), 7.90-7.93 (t, 1H), 7.96-7.99 (d, 1H), 8.21-8.24 (d,1H) .

(d) Acid 1j (2.23 g) was suspended in excess SOCl₂ (35 mL) and thereaction mixture was heated until the acid dissolved and refluxed foranother 30 min. The solvent was removed under reduced pressure and theacid chloride was combined with 2,3,6-trifluorophenol (0.69 g) and 15 mLof CH₂ Cl₂. Pyridine (3.1 mL) was added dropwise under argon. Thesolution was stirred overnight at room temperature for about 4 days. Thevolatiles were removed under reduced pressure. The crude product wassubjected to column chromatography on silica using 50% CH₂ Cl₂ /hexaneto give 1.18 g of ester 3j: 1H NMR (CDCl₃) δ 2.881 (s, 3H), 2.930 (s,3H), 7.09-7.31 (m, 2H), 7.35-7.37 (d, 1H), 7.56-7.58 (d, 1H), 7.65-7.72(q, 1H), 7.80-7.86 (t, 1H), 8.23-8.27 (d, 1H), 8.32-8.35 (d, 1H).

(e) To a slurry of ester 3j (350 mg) and NH₄ Cl (491 mg) in Ar-purged2-propanol (20 mL) was added zinc (600 mg) causing immediatedecolorization of the solution. The colorless solution was stirred atroom temperature for 2 h shielded from light. CH₂ Cl₂ was added to thesolution which was filtered after 30 min. The solvents were removed fromthe filtrate under reduced pressure. The crude material was purified bycolumn chromatography on silica using 5-10% ethyl acetate/hexane toyield 233 mg of acridan 6j: ¹ H NMR (CDCl₃) δ 2.290 (s, 3H), 2.420 (s,3H), 5.504 (s, 1H), 6.141 (s, 1H), 6.73-7.01 (m, 6H), 7.20-7.23 (t, 1H),7.46-7.48 (d, 1H).

(f) The reduced product, acridan 6j (233 mg) was methylated by stirringovernight with methyl triflate (5 mL) under argon with protection fromlight. The volatiles were evaporated and the product purified by columnchromatography on silica using 15% ethyl acetate/hexane to yield 224 mgof 5j: ¹ H NMR (CDCl₃) δ 2.410 (s, 3H), 2.465 (s, 3H), 3.506 (s, 3H),5.276 (s, 1H), 6.78-7.06 (m, 5H), 7.13-7.16 (d, 1H), 7.29-7.38 (m, 2H).

Example 10 Synthesis of Compound 5k

2',3',6'-Trifluorophenyl 1,4-dimethoxy-10-methylacridan-9-carboxylate.

(a) 2,5-Dimethoxyaniline (30 g, Aldrich) was converted to the acetamidederivative by reaction with 24 mL of acetic anhydride, 22.4 mL of aceticacid and 120 mg of zinc at reflux for 3 h. The reaction mixture waspoured into 700 mL of ice-water and stirred. The purple solid wasfiltered, washed with 1 L of water and air-dried yielding 27.3 g of theproduct: ¹ H NMR (CDCl₃) δ 2.200 (s, 3H), 3.781 (s, 3H), 3.842 (s, 3H),6.543-6.584 (dd, 1H), 6.77-6.80 (d, 1H), 7.77 (bs, 1H) 8.10-8.11 (d,1H).

(b) 2,5-Dimethoxyacetanilide (12 g) was condensed with 3-bromoanisole(19.4 mL) in the presence of 8.75 g of K₂ CO₃ and 1.27 g of CuI atreflux for 3 d. After standing overnight, the mixture was heated andthen extracted with toluene (4×100 mL) and evaporated to a brown solid.

The solid was dissolved in 300 mL of ethanol, 6.9 g of KOH were addedand the mixture refluxed for 48 h. The ethanol was evaporated and thecrude product taken up in 300 mL of water and extracted with ethylacetate (3×175 mL). The ethyl acetate extracts were combined, washedwith water, dried over Na₂ SO₄ and evaporated. The crude solid waspurified by passing a CH₂ Cl₂ solution through a short column containinglayered Na₂ SO₄ /silica/Na₂ SO₄ to produce 13.81 g of the diphenylaminecompound: ¹ H NMR (CDCl₃) δ 3.750 (s, 3H), 3.864 (s, 3H), 6.210 (bs,1H), 6.34-6.38 (dd, 1H), 6.79-6.82 (d, 1H), 6.92-7.00 (m, 2H), 7.18-7.20(d, 2H), 7.28-7.33 (t, 2H).

(c) Reaction of 2,5-dimethoxydiphenylamine (5.0 g) with 2.19 mL ofoxalyl chloride in 50 mL of CH₂ Cl₂ and followed by reaction with 5.82 gof AlCl₃ in CH₂ Cl₂ at reflux produced the isatin which was purified byflash chromatography on silica with CH₂ Cl₂ yielding 2.2 g of theproduct as an orange solid: ¹ H NMR (CDCl₃) δ 3.756 (s, 3H), 3.799 (s,3H), 6.59-6.62 (d, 1H), 6.88-6.89 (d, 1H), 6.98-7.05 (m, 2H), 7.11-7.16(t, 1H), 7.48-7.53 (t, 1H), 7.66-7.69 (d, 1H).

(d) The isatin (3.55 g) was converted to1,4-dimethoxy-acridine-9-carboxylic acid (1k) by refluxing with 30 mL of10% KOH and neutralization with 3:1 ice/5M HCl yielding 3.4 g of acid1k: ¹ H NMR (DMSO-d₆) δ 3.934 (s, 3H), 4.018 (s, 3H), 6.99-7.02 (d, 1H),7.21-7.24 (d, 1H), 7.73-7.78 (t, 1H), 7.92-7.99 (m, 2H), 8.27-8.30 (d,1H).

(e) Acid 1k (2.0 g) was suspended in excess SOCl₂ and the reactionmixture was heated until the acid dissolved. The solvent was removedunder reduced pressure and the acid chloride was combined with2,3,6-trifluorophenol (1.05 g) in CH₂ Cl₂. Pyridine (2.5 mL) was addedand the solution was stirred overnight at room temperature under argon.The volatiles were removed under reduced pressure. The crude product wassubjected to column chromatography on silica using CH₂ Cl₂ to give 0.44g of ester 3k: ¹ H NMR (CDCl₃) δ 4.007 (s, 3H), 4.143 (s, 3H), 6.81-6.84(d, 1H), 7.00-7.22 (m, 3H), 7.68-7.73 (dt, 1H), 7.83-7.89 (dt, 1H),8.23-8.26 (d, 1H), 8.45-8.48 (d, 1H).

(f) To a slurry of ester 3k (440 mg) and NH₄ Cl (569 mg) in Ar-purged2-propanol (15 mL) was added zinc (696 mg) causing immediatedecolorization of the solution. The colorless solution was stirred atroom temperature for 2.5 h shielded from light. CH₂ Cl₂ was added to thesolution which was then filtered. The solvents were removed from thefiltrate under reduced pressure. The crude material was again extractedwith CH₂ Cl₂ and the filtrate evaporated. The product was purified bycolumn chromatography using 2.5-20% ethyl acetate/hexane to yield 272 mgof acridan 6i: ¹ H NMR (CDCl₃) δ 3.836 (s, 3H), 3.874 (s, 3H), 5.601 (s,1H), 6.31-6.34 (d, 1H), 6.68-7.01 (m, 6H), 7.17-7.22 (t, 1H), 7.51-7.54(d, 1H).

(g) The reduced product, acridan 6k (272 mg) was methylated with methyltriflate (0.74 mL) in 5 mL of CH₂ Cl₂ under argon with protection fromlight. After 3 d the volatiles were evaporated and the product purifiedby column chromatography using 5% ethyl acetate/hexane yielding 200 mgof 5k: ¹ H NMR (CDCl₃) δ 3.581 (s, 3H), 3.817 (s, 3H), 3.866 (s, 3H),5.547 (s, 1H), 6.50-6.53 (s, 1H), 6.77-7.01 (m, 4H), 7.08-7.11 (d, 1H),7.27-7.33 (m, 1H), 7.40-7.43 (d, 1H).

Chemiluminescence Measurements

The experiments in the following examples were performed using either aTurner Designs TD-20e (Sunnyvale, Calif.) luminometer fitted withneutral density filter for light attenuation or a Labsystems Luminoskan(Helsinki, Finland) luminometer. Data collection, analysis and displaywere software controlled.

Example 11

A detection reagent was prepared by combining in a 40:1 ratio reagent Aconsisting of: 0.6 mM urea peroxide, 0.1 mM p-phenylphenol, 0.025% TWEEN20, 1 mM EDTA in 0.01M tris buffer, pH 8.0 and reagent B consisting of:acridan 5c (0.86 mg/mL) in 1:1 (v/v) p-dioxane/ethanol or 1:1 (v/v)propylene glycol/ethanol. To 40 μL of the resulting solution, 1 μL ofHRP ((1.4×10⁻¹⁶ mol)) was added and the solution incubated for 5 min. Aflash of luminescence was induced by injecting 100 μL of 0.1M NaOHsolution. A blank was performed by repeating the experiment without theaddition of enzyme. FIG. 1 shows the generation of light emission whichresulted.

Example 12

An experiment according to Example 11 was repeated using a detectionreagent prepared by combining reagents A and B in a 1200:1 ratio and anincubation time of 100 sec. A better signal/background ratio resulteddue to a lowering of the light intensity of the blank.

Example 13

The sensitivity and linearity of detection of HRP using the detectionreagent of Example 12 was determined. In each of 3 wells of amicroplate, 50 μL volumes of the detection reagent were mixed at roomtemperature with 1 μL aliquots of solutions of HRP containing between1.4×10⁻¹⁵ and 1.4×10⁻¹⁹ mol of enzyme. After 100 sec, 100 μL of 0.1MNaOH was added and luminescence integrated for 2 sec. FIG. 2 shows thelinear range of HRP amount measured using a reagent of the presentinvention containing acridan 5c.

Example 14

A detection reagent according to the composition of Example 11containing instead the acridan 5b was tested for detection of HRP. Themethod specified in example 10 was followed with the exception that thedetection solution was incubated with the enzyme for 5 min. The lowestdetected amount of HRP was 1.4 amol (1.4×10⁻¹⁸ mol) with asignal/background ratio of 2.

Example 15

A detection reagent according to the composition of Example 11containing instead a slightly impure preparation of the acridan 5d wastested for detection of HRP. Following the method specified in example10, 1.4×10⁻⁶ mol of HRP incubated with the reagent and flashed with NaOHproduced a signal 105 times greater than the blank. Using 1.4×10⁻¹⁷ molof HRP and incubating for 10 min produced a signal 69 times greater thanthe blank. The principal impurity, the N-demethylated analog (6d) wastested independently under the conditions of the experiment and foundnot to produce a significant amount of light.

Example 16

A detection reagent according to the composition of Example 11containing instead a crude preparation of the acridan 5h was tested fordetection of HRP. Following the method specified in example 10,1.4×10⁻¹⁶ mol of HRP incubated with the reagent for 3 min and treatedwith 0.1M NaOH produced a signal 72 times greater than the blank. Theprincipal impurity, the N-demethylated analog (6h) was testedindependently under the conditions of the experiment and found not toproduce light.

Example 17 Effect of Enhancers

Detection reagent solutions according to the composition of Example 11may be prepared with substitution of various phenolic enhancers, reactedwith HRP and subsequently made highly basic. Useful levels of lightintensity compared to reagent background are obtained with reagentsincorporating p-iodophenol, p-bromophenol, p-hydroxycinnamic acid,2-naphthol, 6-bromo-2-naphthol and 4-iodophenylboronic acid.

Example 18 Effect of Peroxide

Detection reagent solutions according to the compositions of Example 11may be prepared with substitution of various peroxides, reacted with HRPand subsequently made highly basic. Useful levels of light intensitycompared to reagent background are obtained with reagents incorporatinghydrogen peroxide, sodium perborate and urea peroxide.

Example 19

A solution of the reagent of Example 11 containing acridan 5c (3 mL) wasplaced in a quartz cuvette in a Varian Cary 3E (Palo Alto, Calif.)UV-Vis spectrophotometer. HRP (1.4×10⁻¹⁵ mol) was added and theabsorbance spectrum between 300-500 nm scanned at 30 sec intervals. FIG.3 shows the formation (in the direction bottom curve to top curve at 400nm) of the acridinium compound 4c. After 15 min no further change wasobserved in the spectrum. A 0.1M NaOH solution was added causing a burstof blue light. The spectrum of the resulting solution matched theabsorption of 1,6-dimethoxy-10-methylacridone.

Example 20

To demonstrate that acridans bearing alkyl, alkoxy and/or halogensubstituents on the acridan ring provide superior performance to similaracridans which are unsubstituted on the acridan ring, the experiment ofExample 20 was repeated using an acridan compound which is unsubstitutedon the acridan ring. A solution of a reagent similar to that used inExample 11 (3 mL) but containing the acridan 2',6'-difluorophenyl10-methylacridan-9-carboxylate was reacted with HRP (1.4×10⁻¹⁵ mol) andthe absorbance spectrum between 300-500 nm scanned at 30 sec intervals.FIG. 4 shows the formation (in the direction bottom curve to top curveat 400 nm) of both the acridinium compound 2',6'-difluorophenyl10-methylacridinium-9-carboxylate and N-methylacridone as proven bycomparison with authentic samples of these two compounds. In addition,subjecting the acridinium compound 2',6'-difluorophenyl10-methylacridinium-9-carboxylate to the same reagent formulation in theabsence of added HRP led to measurable conversion to the same acridonewithin minutes.

Example 21

The sensitivity and linearity of detection of HRP using the detectionreagent containing acridan 5i was determined. The reagent containedacridan 5i (0.05 mM) (diluted 1:40 from a 2 mM stock solution in 1:1ethanol/p-dioxane), 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 0.5 mM ureaperoxide and 1 mM EDTA in 0.01M tris buffer, pH 8.0. In each of 3 wellsof a microplate, 100 μL volumes of the detection reagent were mixed atroom temperature with 10 μL aliquots of solutions of HRP containingbetween 1.4×10⁻¹⁵ and 1.4×10⁻¹⁹ mol of enzyme or 10 μL of water as areagent blank. After 5 min, 100 μL of 0.1M NaOH was added and the totalluminescence integrated for 2 sec. Light emission was proportional tothe amount of HRP with a calculated detection limit <10⁻¹⁸ mol.

Example 22

The sensitivity and linearity of detection of HRP using the detectionreagent containing acridan 5j was determined. The reagent containedacridan 5j (0.05 mM) (diluted 1:40 from a 2 mM stock solution in 1:1ethanol/p-dioxane), 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 0.5 mM ureaperoxide and 1 mM EDTA in 0.01M tris buffer, pH 8.0. In each of 3 wellsof a microplate, 100 μL volumes of the detection reagent were mixed atroom temperature with 10 μL aliquots of solutions of HRP containingbetween 1.4×10⁻¹⁵ and 1.4×10⁻¹⁹ mol of enzyme or 10 μL of water as areagent blank. After 5 min, 100 μL of 0.1M NaOH was added and the totalluminescence integrated for 2 sec. Light emission was proportional tothe amount of HRP with a calculated detection limit of 1.4×10⁻¹⁹ mol.Similar results were obtained in another set of experiments in which theflash reagent consisted of 0.1M NaOH and 0.1 mM SDS and peak lightintensity was measured instead of total intensity.

Example 23

The sensitivity and linearity of detection of HRP using the detectionreagent containing acridan 5k was determined. The reagent containedacridan 5k (0.05 mM) (diluted 1:40 from a 2 mM stock solution in 1:1ethanol/p-dioxane), 0.1 mM p-phenylphenol, 0.025% TWEEN 20, 0.5 mM ureaperoxide and 1 mM EDTA in 0.01M tris buffer, pH 8.0. In each of 3 wellsof a microplate, 100 μL volumes of the detection reagent were mixed atroom temperature with 10 μL aliquots of solutions of HRP containingbetween 1.4×10⁻¹⁵ and 1.4×10⁻¹⁹ mol of enzyme or 10 μL of water as areagent blank. After 5 min, 100 μL of 0.1M NaOH was added and themaximum luminescence intensity was measured. Light intensity wasproportional to the amount of HRP with a calculated detection limit<10⁻¹⁸ mol.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe appended claims.

We claim:
 1. A compound which is 2',3',6'-trifluorophenyl1,6-dimethoxy-10-methylacridan-9-carboxylate.
 2. A compound which is2',3',6'-trifluorophenyl4-chloro-3-methoxy-10-methylacridan-9-carboxylate.
 3. A compound whichis 4'-fluorophenyl4-chloro-3-methoxy-10-methylacridan-9-thiocarboxylate.
 4. A compoundwhich is2',3',6'-trifluorophenyl-1,4-dimethoxy-10-methylacridan-9-carboxylate.5. A compound which is 2',3',6'-trifluorophenyl1,4,10-trimethylacridan-9-carboxylate.
 6. A compound which is2',3',6'-trifluorophenyl1,6-dimethoxy-4,10-dimethylacridan-9-thiocarboxylate.
 7. A compoundhaving the formula: ##STR11## wherein X is an anion.
 8. A compoundhaving the formula: ##STR12## wherein X is an anion.
 9. A compoundhaving the formula: ##STR13## wherein X is an anion.
 10. A compoundhaving the formula: ##STR14## wherein X is an anion.
 11. A compoundhaving the formula: ##STR15## wherein X is an anion.
 12. A compoundhaving the formula: ##STR16## wherein X is an anion.